{62 -carboxyacrylamidine

ABSTRACT

New compounds cis- Beta -carboxyacryl-amidine and trans- Beta carboxyacrylamidine and processes for preparing the same. These compounds can be used as antifungal agents against Blastomyces dermatitides and Coccidioides immitis.

United States Patent [72] inventors Edward G. Daniels Portage;

Le Roy E. Johnson, Kalamazoo Township, Kalamazoo County; Floyd P.Kupiecki, Portage; Paul F. Wiley, Texas Township, Kalamazoo County, allof Mich. 484,086

Aug. 31, 1965 Dec. 7, 197 1 The Upjohn Company Kalamazoo, Mich.

[21] Appl. No. [22] Filed [45] Patented [73] Assignee [54]B-CARBOXYACRYLAMIDINE 12 Claims, No Drawings 1521 U,S.Cl 260/53411,195/47, 260/253, 260/295, 260/295.5,

51 ll'lLCl ..C07c 123/00 [50] Field of Search 260/534, 501

[56] References Cited UNITED STATES PATENTS 2,85 1 ,490 9/1958 066:, Jr.et al. 260/534 2,955,136 10/1960 Sullivan et all. 260/537 OTHERREFERENCES Katsev et al., Chemical Abstracts Vol. 56: 12,727, 1962)Primary Examiner- Lorraine A. Weinberger Assistant Exam iner-RobertGerstl A110rneyGeorge T. Johannesen ABSTRACT: New compoundscis-B-carboxyacryl-amidine and trans-B-carboxyacrylamidine and processesfor preparing the same. These compounds can be used as antifungal agentsagainst Blaslomyces dermatitides and Coccidiuides immitis.

B-CARBOXYACRYLAMIDINE This invention relates to novel compositions ofmatter and to processes for the'production thereof. More particularly,This invention relates to the new compounds cis-B-carboxyacrylamidine(l) and trans-B-carboxyacrylamidine (II) and to processes for theproduction thereof.

The novel compounds of this invention have the following structuralformulas:

II C THE MlCRO-ORGANISM TABLE 1 Appearance 0 Ektachrome Agar Medium S.furlongus v. furlongus Bennett's Gray aerial growth.

Brown reverse. Czapek's Sucrose Gray aerial growth. Pale brown reverse.Maltose Tryptone Blue-gray aerial growth.

Brown reverse. No aerial growth. Brown reverse. Melanin Peptone-Iron0.1% Tyrosine Gray aerial growth.

Brown reverse. Casein Starch Gray-blue aerial growth.

Brown reverse.

'Dietz, A. Ektachrome Transparencies as Aids in ActinomyceteClassification," AnnaLr ufrhe N. Y. Academy afSciencer 60: I52-l54,1954.

TABLE n Assimilation of Carbon Compounds in Synthetic Medium (J. Bact.56: 107-1 14, 1948) S. furlongu: v. furlongus Control 2. L-arabinose 3.rhamnose 4. D'fructose S. D-galactose 6. D-glucose 7. D-mannoae B.maltose 9. sucrose 10. lactose l l. cellobiose l3. dextrin l4. inulinl5. soluble starch l6. glycerol l7. dulcitol l8. D-mannitol l9.D-sorbitol 20. inositol 2l. salicin 22. phenol 23. cresol 24. Na formate25. Na oxalate 26. Na tartrate 27. Na salicylate 28. Na acetate 29. Nacitrate 30. Na succinate positive assimilation positiveassimilationslight growth slight growth-no assimilation Pcptone-ironAgar Blue-gray aerial growth.

Brnwm reverse.

Brown pigment.

Melanin positive.

Trace gray-white aerial growth.

Colorless reverse.

No pigment.

Malate not solubilized.

Gray pink aerial growth.

Browntan reverse.

Tan pigment.

Gray-pink aerial growth on periphery.

Brown-tan reverse.

Tan pigment.

Casein not (or very slightly) solubilized under growth.

Gray-pink aerial growth.

Brown reverse.

Brown pigment.

Tyrosine solubilized.

Pale gray-pink aerial growth.

Pale yellow-tan reverse.

Pale yellow-tan pigment.

Xanthine not solubilized.

Pale gray-pink aerial growth.

Yellow reverse,

Yellow pigment.

Starch hydrolyzed.

Blue-gray aerial growth Brown reverse.

Tan pigment.

Calcium Malate Agar Glucose Asparagine Agar Skim Milk Agar Tyrosine AgarXanthine Agar Nutrient Starch Agar Yeast Extract- Malt Extract AgarBennett's Agar Czapek's Sucrose Agar 24 C. 23 C. 37 C. 55 C.

Maltose-Tryptone Agar Litmus Milk Plain Gelatin Nutrient GelatinNutrient Nitrate Broth Synthetic Nitrate Broth Heavy gray aerial growthon periphery.

Tan reverse.

Tan pigment.

Heavy gray with trace blue aerial growth.

Olive-tan reverse.

Olive-tan pigment.

Good blue-gray aerial growth Tan-brown reverse.

Tan-brown pigment.

Heavy gray aerial growth. Red-tan reverse.

Red-tan pigment.

Good vegetative growth at l8 hours.

Fair gray aerial growth.

Gray reverse.

No pigment.

same as at l8 C.

Same as at l8 C.

Same as at l8 C.

Good vegetative growth at l8 hours.

Cream-tan aerial growth Tan reverse.

Tan pigment.

Heavy gray-blue aerial growth. Blue-tan reverse.

Tan pigment.

Heavy blue-gray aerial growth. Blue-brown reverse.

Tan pigment.

Heavy gray aerial growth flecked with tan Red-tan reverse.

Red-tan pigment.

Good vegetative growth at l8 hours.

Gray aerial growth on surface ring.

Peptonization.

Reduction.

White aerial growth on surface.

TABLE IV Agar Medium 8. furlongu: v. furlangus Color Harmony Manual 3rdEd.

lSCC-NBS Color Names Bennett's Surface a white 15" light blue, babyblue, light horizon blue, light sky blue Jig beige brown, mist brownReverse 3rrg yellow maple 77m moderate yellowiah brown Pigment 2pg(nr)mustard gold 943m light olive brown Cupeks Sucrose Surface 23c bamboo.chamoia m grayish yellow Reverse Zec biscuit, ecru, 903m grayish yellowoatmeal sand Pigment none Maltose- 'l'ryptone Surface c light gray 264gmlight gray l5ca pale blue Reverse Bpi golden brown, 75g deep yellowishtobacco brown brown 77m moderate yellowish brown Pigment 3n: topaz,butter- 72m dark orange yellow scotch 74g strong yellowish brown TABLE VMicroscopic Characteristic S. furlangur v. furlongru Light Microscope RFshort Electron Microscope Direct Irregular, beadlike Smooth.

Carbon Replica Irregular with surface ridging.

cis-B-Carboxyacrylamidine (l) is produced when the elaborating organismis grown in an aqueous nutrient medium under submerged aerobicconditions; for the preparation of limited amounts, surface cultures inbottles can be employed. The organism is grown in a nutrient mediumcontaining a carbon source, for example, an assimilable carbohydrate,and a nitrogen source, for example, an assimilable nitrogen compound orproteinaceous material. Preferred carbon sources include glucose, brownsugar, sucrose, glycerol, starch, corn starch, galactose, dextrin,molasses, and the like. Preferred nitrogen sources include com steepliquor, yeast, autolyzed brewers yeast with milk solids, soybean meal,cottonseed meal, corn meal, milk solids, pancreatic digest of casein,distillers solubles, fish meal, animal peptone liquors, meat and bonescraps, and the like. A combination of these carbon and nitrogen sourcescan be used advantageously. Trace metals, for example, zinc, magnesium,manganese, cobalt, iron, and the like need not be added to thefermentation since tap water and unpurified ingredients are used asmedia components.

Production of the compound of the invention can be effected at anytemperature conducive to satisfactory growth of the micro-organism forexample, between about l8 and 40 C. and preferably between about 25 and30 C. Ordinarily, optimum production of the compound is obtained inabout 2 to 10 days. The medium normally stays fairly close to neutral,or on the acid side during the fermentation. The final pH is dependent,in part, on the buffers present, if any, and in part on the initial pHof the culture medium which is advantageously adjusted to about pH 6-8prior to sterilization.

When growth is carried out in large vessels and tanks, it is preferableto use the vegetative form, rather than the spore form, of themicro-organism for inoculation to avoid a pronounced lag in theproduction of the new compound and the attendant inefficient utilizationof equipment. Ac-

cordingly, it is desirable to produce a vegetative inoculum in anutrient broth culture by inoculating the broth culture with an aliquotfrom a soil or slant culture. When a young, active, vegetative inoculumhas thus been secured, it is transferred aseptically to large vessels ortanks. The medium in which the vegetative inoculum is produced can bethe same as, or different from, that utilized for the production of thenew compound, as long as it is such thata good growth of themicro-organism is obtained.

cis-B-Carboxyacrylamidine (I) is an amphoteric compound having theempirical formula C,H N,O It is relatively insoluble in the commonorganic solvents, e.g., amyl acetate, butanol, methylene chloride;slightly soluble in hot water; and readily soluble in (MN hydrochloricacid and 0.1N sodium hydroxide.

cis-fi-Carboxyacrylamidine (I) can be recovered from the culture mediumby the use of absorptive techniques. In a preferred process,cis-B-carboxyacrylamidine (I) is recovered from its culture medium byseparation of the mycelium and undissolved solids by conventional meanssuch as by filtration or centrifugation. The antibiotic is then removedfrom the filtered or centrifuged broth by the use of activated carbon.After the antibiotic is adsorbed on the carbon, acetone can be used asthe eluting agent. The eluates obtained from the carbon column can beevaporated to dryness to provide an impure preparation ofcis-fi-carboxyacrylamidine (I). This preparation can be used inenvironments where higher purity of cis-B-carboxyacrylamidine (I) is notnecessary.

High purity cis-B-carboxyacrylamidine (I) can be obtained by subjectingan impure dry preparation of cis-B-carboxyacrylamidine (I), as obtainedabove, to further absorption on an activated carbon column. Elution ofthe antibiotic from this second carbon column absorption can beaccomplished by the use of varying concentrations of acetone. Theeluates from the column can be concentrated under reduced pressure atwhich time the cis-B-carboxyacrylamidine (I) crystallizes.

Since cis-B-carboxyacrylamidine (I) and trans-B-carboxyacrylamidinc (II)are amphoteric substances, they form salts with acids, alkali metals,alkaline earth metals, and amines. Metal salts can be prepared bydissolving cis-B-carboxyacrylamidine (l) or transB-carboxyacrylamidine(II) in water, adding a dilute metal base until the pH of the solutionis about 7 to 8, and freeze-drying the solution to provide a driedresidue of the cis-B-carboxyacrylamidine (I) ortransB-carboxyacrylamidine (ll) metal salt. Such metal salts include thesodium, potassium, and calcium salts. Amine salts, including those withorganic bases, such as primary, secondary, and tertiary mono-, di-, andpolyamines can also be formed using the above-described or othercommonly employed procedures. Other salts are obtained withtherapeutically effective bases which impart additional therapeuticeffects thereto. Such bases are, for example, the purine bases such astheophylline, theobromine, caffeine, or derivatives of such purinebases; antihistaminic bases which are capable of forming salts with weakacids; pyridine compounds such as nicotinic acid amide, isonicotinicacid hydrazide, and the like; phenylalkylamines such as adrenalin,ephedrine, and the like; choline, and others.

Acid salts of cis-B-carboxyacrylamidine (I) ortrans-fi-carboxyacrylamidine (II) can be made by neutralization with theappropriate acid to below about pH 7.0, and advantageously to about pH 2to pH 6. Suitable acids for this purpose include hydrochloric, sulfuric,phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, pamoic,cholic, palmitic, mucic, camphoric, glutaric, glycolic, phthalic,tartaric, lauric, stearic, salicylic, 3phenylsalicylic,5-phenyl-salicylic, 3-methylglutaric, or'tho-sulfobenzoic,cyclohexanesulfamic, cyclopentanepropionic, l,2-cyclohexanedicarboxylic, 4-cyclohexenecarboxylic,octadecenylsuccinic, octenylsuccinic,

methanesulfonic, benzenesulfonic, helianthic, Reineckes,dimethyldithiocarbamic, sorbic, monochloracetic, undecylenic,4'-hydroxyazobenzene-4-sulfonic, octadecylsulfuric, picric, benzoic,cinnamic, and like acids.

trans-B-Carboxyacrylamidine (II) is produced upon acidic treatment ofcis-B-carboxyacrylamidine (I) with a strong mineral acid, i.e., l to 6 Nacid. Acidic treatment of cis-B-carboxyacrylamidine (l) at roomtemperature gives trans-B-carboxyacrylamidine (II) which is useful inthe same manner as cis-B-carboxyacrylamidine (I). The isomerizationreaction can be effected, advantageously, with a mineral acid, forexample, hydrochloric acid, sulfuric acid, and the like. Heat can beemployed, advantageously, to reduce the reaction time. The isomerizationproduct can be recovered from the reaction mixture by adjusting thereaction mixture to a pH of about 4-5 with a base, for example, sodiumhydroxide, and then allowing the crystalline precipitate oftrans-B-carboxyacrylamidine (II) to form.

The novel compounds of the invention have antifungal activity againstthe following fungi:

Blastomyces dermatitides Coccidioides immitis Hormadendrum compactumNocardia asteroides Geotrichium sp.

Cryptococcus neoformans Sporotrichum schenkii Trichophyton rubrumMicrasporum canis Candida albicans (Abbott) Trichaphyton vialaceumTrichophyton asteroides Trichophyton mentagraphytes Thus, since thenovel compounds of the invention are active against C. neoformans, theycan be used to treat pigeon roosts to inhibit this fungus which has beenfound in pigeon droppings. (Journal of The American Medical Association,Vol. 191, No.4, Jan. 25, l965, pp. 269-274.) The novel compounds of theinvention can also be used as the antifungal agents in the shoe uppersdisclosed in U.S. Pat. No. 3,l 30,505. Furthermore, the novel compoundsof the invention can be used to swab laboratory benches and equipment ina mycological laboratory.

The new compounds of the invention are active against KB cells in tissueculture and, thus, they can be used for washing surgeons gloves andinstruments during cancer operations. They can also be used to destroythe surface of ulcerating tumors.

The following examples are illustrative of the process and products ofthe present invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE I A. Fermentation A soil stock of Streptomycesfurlongus var.furlongus, NRRL 3192, was used to inoculate 500-ml. Erlenmeyer flaskscontaining ml. of sterile seed medium consisting of the followingingredients:

Glucose monohydrate 25 g. Pharmamedia 25 g. Tap water q.s. l l.

Pharmamedia is an industrial grade of cottonseed lz'lour produced byTrader's Oil Mill Company, Fort Worth, Texas.

The seed medium presterilization pH was 7.2. The seed inoculum was grownfor 2 days at 28 C. on a Gump rotary shaker operating at 250 rpm.

The seed inoculum (5 ml.) was used to inoculate a 500-ml. Erlenmeyerfermentation flask containing 100 ml. of the following sterilefermentation medium:

Glucose monohydrate 20 g.ll. ltaysoy l0 g./l. Pabst yeast 2.5 5.1].Ammonium sulfate (NH,),SO 5 3.". KCL 3 g.ll. Calcium carbonate 4 g./l.

Tapwater Balance 'Kaysoy is a fat-extracted, finely milled, soybeanmeal.

The presterilization pH of the fermentation flask was 6.8. Thefermentation cycle was 4 days during which time the flask was grown withincubation at 28 C. on a Gump rotary shaker operating at 250 r.p.m. Thepreharvest beer assayed 8 biounits/ml. on the KB cells in agar assay.The KB cells in agar assay procedure is as follows:

KB cells are grown in tissue culture and the cells are harvested andpooled every 48 hours. These pooled cells are uniformly dispersed intoan agar-serum mixture at 45 C. and the agar mixture is poured rapidlyinto chromatography trays to about a 2-3 mm. depth. After the agarsolidifies, dried paper discs, which were previously saturated withdimethylformamide solutions of the test compounds, are applied to theagar surface and the trays are inverted and incubated at 37 C. After 16hours the agar surface is sprayed with a redox dye,sodium-2,6-dichlorophenol indophenol (DCIP), prepared in isotonicsaline, and the cells are incubated for approximately 30 minutes at 37C. 1n areas where the cells are either killed or severely inhibited bythe material under test, the dye is at best only poorly reduced and ablue zone of inhibition is obtained. Where the cells are uneffected,good dye reduction is obtained and a colorless background is observed.

B. Carbon Absorption The whole broth (500 liters assaying 13biounits/ml.) from a scaled-up fermentation, as described above, wasslurried with percent of its weight of diatomaceous earth, filtered, andthe cake washed with water. The filtered beer and wash (605 liters) wasadjusted to pH 3.0 with 6 N hydrochloric acid and passed through acolumn containing activated carbon. The carbon column was eluted with a50 percent aqueous acetone solution. The aqueous acetone eluate (480liters) was concentrated to an aqueous liters) and freeze-dried to give1903 g. of an impure preparation of cis-B-carboxyacrylamidine (l)assaying 1.2 biounits/mg. on the KB cells in agar assay.

C. Second Carbon Column Absorption Nine hundred grams (900 g.) ofcis-B-carboxyacrylamidine (1), obtained as above, was dissolved in waterto a concentration of 30 mg./ml. The pH was adjusted to 5.0 withhydrochloric acid and the solution filtered to remove undissolvedmaterial. The filtrate was then passed through a column containingactivated carbon 1 .3 g. of carbon per g. of product) at a rate of 2 to2.5 ml./gram of carbon/hour. The column was washed with deionized water(4 ml. of deionized water/gram of carbon). The carbon column was theneluted with the following eluants:

3 percent acetone in water (8 mL/gram of carbon) 12 percent acetone inwater l5 m1./gram of carbon) 25 percent acetone in water (8 ml./gram ofcarbon) Fractions, approximately equal to the column holdup volume, werecollected, weighed, and assayed for bioactivity on the KB cells in agarassay. Fractions containing the highest specific gravity (usually in the12 percent acetone eluate) were pooled and concentrated under reducedpressure to about 5 percent of the original volume at which time thecis-B-carboxyacrylamidine (l) crystallized from the concentrate. Coolingand further concentration'of the solution yielded additional crystallinecis-fl-carboxyacrylamidine (I). The crystalline product was washed firstwith cold water, then with cold ethanol, and finally air dried. From the900 g. of starting material there was obtained 26.9 g. of crystallinecis-B-carboxyacrylamidine (l) assaying 13.7 biounits on the KB cells inagar assay.

D. Characterization of cis-B-carboxyacrylamidine (l) Melting point:223-23 1 C.

Elemental analysis: Calcd. for C H N,O C, 42.10; H,

Found: C, 41.93; H, 5.49; N, 23.88; 0, 27.59.

Color: Crystals are colorless.

Optical activity: None Ultraviolet absorption spectrum: Maximum at 211mg with e 10,550. (In H O).

lnfrared absorption spectrum: lnfrared spectrum in mineral oil mullshows major absorption bands at the following frequencies: 3,060, 1,725,1,680 sh, 1,620, 1,580, 1,530, 1,510, 1,485, 1,335, 1,190, 885, 810,775, and 755 cm..

Hydrolysis product: Alkaline hydrolysis with 1 N sodium hydroxidesolution releases ammonia and maleic acid.

EXAMPLE 2 trans-fi-Carboxyacrylamidine One gram (1 g.) ofcis-B-carboxyacrylamidine, prepared as in example 1, in 25 ml. of 6 Nhydrochloric acid, was heated on the steam bath for 6 hours. Thereaction mixture was adjusted to pH 4.5 with a 4 N sodium hydroxidesolution and allowed to stand overnight. The crystalline precipitate oftrans- B-carboxyacrylamidine (11) was recovered by filtration; yield 464mg. The characteristics of this compound are as follows:

Elemental analysis: Calcd. for C H,N,O C, 42.10; H, 5.68;

Found: C, 41.78; H, 5.71; N, 23.57.

Melting point: Decomposes above 280 C. without noticeable gas evolution.

lnfrared absorption spectrum: The isomerization product when suspendedin mineral oil mull gave the following major bands: 730, 755, 780,1,145, 1,375, 1,505, 1,555, 1,600, 1,730, 2,680, 3,140 cmf.

Hydrolysis product: Alkaline hydrolysis on a steam bath with 1 N sodiumhydroxide solution releases ammonia and fumaric acid.

Ultraviolet absorption spectrum: Maximum at 213 mg, with e 11,514(ln HO).

The reaction mixture disclosed in example 2 contained cisandtrans-Bcarboxyacrylamidine. Mixtures of cisand trans-B-carboxyacrylamidine can be used for the same purposes as thecomponents in environments where separation of the components is notadvantageous.

We claim:

1. A compound selected from the class consisting of cis-B-carboxyacrylamidine of the formula:

and pharmaceutically acceptable salts thereof.

2. cis-B-Carboxyacrylamidine.

3. A compound selected from the group consisting of cis-B-carboxyacrylamidine, and pharmaceutically acceptable salts thereof withalkali metals, alkaline earth metals, and amines.

4. A compound selected from the group consisting of cis-B-carboxyacrylamidine, according to claim 1, and pharmaceuti callyacceptable acid addition salts thereof.

5. A compound, as defined in claim 1, cis-B-carboxyacrylamidine, in itscrystalline form.

6. A compound selected from the class consisting of trans-B-carboxyacrylamidine of the formula:

tically acceptable acid addition salts thereof.

10. A compound, as defined in claim 6, trans-B-carboxyacrylamidine, inits crystalline form.

111. A compound selected from the class consisting of cisB-carboxyacrylamidine, trans-B-carboxyacryiamidine, and pharmaceuticallyacceptable salts thereof.

12. A composition of matter consisting of cis-B-carboxyacrylamidine andtrans-B-carboxyacrylamidine.

. amass

2. cis- Beta -Carboxyacrylamidine.
 3. A compound selected from the groupconsisting of cis- Beta -carboxyacrylamidine, and pharmaceuticallyacceptable salts thereof with alkali metals, alkaline earth metals, andamines.
 4. A compound selected from the group consisting of cis- Beta-carboxyacrylamidine, according to claim 1, and pharmaceuticallyacceptable acid addition salts thereof.
 5. A compound, as defined inclaim 1, cis- Beta -carboxyacrylamidine, in its crystalline form.
 6. Acompound selected from the class consisting of trans- Beta-carboxyacrylamidine of the formula: and pharmaceutically acceptablesalts thereof.
 7. trans- Beta -Carboxyacrylamidine.
 8. A compoundselected from the group consisting of trans- Beta -carboxyacrylamidine,and pharmaceutically acceptable salts thereof with alkali metals,alkaline earth metals, and amines.
 9. A compound selected from the groupconsisting of trans- Beta -carboxyacrylamidine, according to claim 6,and pharmaceutically acceptable acid addition salts thereof.
 10. Acompound, as defined in claim 6, trans- Beta -carboxyacrylamidine, inits crystalline form.
 11. A compound selected from the class consistingof cis- Beta -carboxyacrylamidine, trans- Beta -carboxyacrylamidine, andpharmaceutically acceptable salts thereof.
 12. A composition of matterconsisting of cis- Beta -carboxyacrylamidine and trans- Beta-carboxyacrylamidine.